Summary Technical Innovation: We have identified a series of specific, well-characterized biomarkers that adipocytes use to regulate energy balance (BCAA?s), appetite (leptin) and inflammation (adipokines). Assays for each of these analytes will be developed with temporal resolutions <1 min, allowing fast changes in uptake or secretion to be measured in response to acute environmental stimuli for the first time. Technologies that will be developed include: online microdialysis-capillary electrophoresis (MD- CE), online aptamer-micro free flow electrophoresis (FFE) affinity assays and online FFE-MALDI. Biological Hypothesis: The assays developed in this proposal will be used to test the hypothesis that artificial sweeteners affect adipocyte metabolism and signaling. The safety of artificial sweeteners is based on the presumption that they are metabolically inert. Any observed changes in BCAA, leptin or adipokine uptake/release would indicate that artificial sweeteners have a direct effect on adipocyte metabolism, appetite control and/or immune response. Long Term Biomedical Significance: The assays developed in this proposal will allow the acute effect of food additives or other stimuli on adipocyte regulation of energy balance, appetite and inflammation to be measured for the first time. These assays will provide valuable information for both assessing the safety of the food supply as well as achieving a better understanding of how adipose tissue regulates these important functions. Together these data may provide key insights into the origins of metabolic disorders and suggest strategies for breaking the cycle of rising obesity levels that is quickly becoming a worldwide epidemic.